STED and GSD fluorescence light microscopy are known as methods of providing a spatial resolution surpassing the diffraction limit in imaging a structure in a sample marked with a fluorescent dye. In STED fluorescence light microscopy, a measurement volume out of which the fluorescence light is spontaneously emitted from the sample is reduced as compared to the diffraction-limited dimensions of a focused beam of excitation light in that the intensity distribution of the excitation light beam in the sample is superimposed with an intensity distribution of stimulation light. The intensity distribution of the stimulation light has a zero point at a measurement point, and adjacent to the measurement point it has such a high intensity that the fluorescent dye is transferred by stimulated emission out of its electronically excited fluorescent state into an energetically lower non-fluorescent electronic state. The measurement point is thus framed by an area out of which no spontaneously emitted fluorescence light may origin due to the intensity of the stimulation light.
In GSD fluorescence light microscopy, the spatial limitation of the area of the sample out of which the spontaneously emitted fluorescence light may origin is achieved in that the fluorescent dye outside the measurement point is transferred into a dark state either directly out of its ground state or out of an electronically excited fluorescent state by means of an optical signal. The fluorescent dye does not get back out of this dark state at least as long as the same measurement point is measured so that no excitation of the fluorescent dye for a spontaneous emission of fluorescence light is possible due to the depletion of the ground state of the fluorescent dye. Here, the intensity distribution of the optical signal depleting the ground state of the fluorescent dye is basically the same as that one of the stimulation light in STED fluorescence light microscopy. This means that when scanning a sample with the actual measurement point each molecule of the fluorescent dye—prior to being reached by the measurement point—is subjected to relevant intensities of the excitation light and of the stimulation light or the optical signal used for depleting the ground state of the fluorescent dye, and has thus already undergone a number of transfer cycles. This number of transfer cycles is associated with a considerable danger of at least temporarily bleaching the fluorescent dye. This danger may be reduced by interrupting the respective optical signal. This, however, elongates the duration of the measurement. Although a lot of even very sensitive fluorescent dyes may return out of their dark state into their ground state excitable for fluorescence at least a few times, they are not suited for high spatial resolution STED and GSD fluorescence light microscopy for the above reasons.
In so-called RESOLFT fluorescence light microscopy, switchable fluorescent dyes are used which are transferable between conformation states with different fluorescence properties. These switchable fluorescent dyes allow to work with lower high light intensities and thus reduce the danger of bleaching the fluorescence dye when applying the principles of STED and GSD fluorescence light microscopy. The conformation states of the switchable fluorescent dyes display a longer lifetime than the electronic states which are the only states usable with simple fluorescent dyes in STED and GSD fluorescence light microscopy. The general problem that an individual molecule of the fluorescent dye has already been subjected to considerable light intensities and thus to a plurality of changes of state within short term prior to be reached by the actual measurement point, however, is still present. Additionally, the selection of switchable fluorescent dyes is still limited despite considerable development efforts, and even many of the available switchable fluorescent dyes are not as often switchable without damages as needed in STED or GSD fluorescence light microscopy when the measurement point gets closer to an individual dye molecule. Thus, the number of possible changes of state of the fluorescent dyes within short term is the limiting factor with all methods of high resolution light scanning microscopy known up to now (see Hell, S. W., “Microscopy and its focal switch”, Nature Methods, Vo. 6 (2009), page 28, right column, §4).
Besides the methods providing a spatial resolution overcoming the diffraction limit in imaging a structure marked with a fluorescent dye in a sample in which the spatial area of the sample out of which the measurement signal may origin is limited, there are methods known as PALM and STORM in which purposefully only a small fraction of the molecules of the substance marking the structure is activated into an active state and excited in this active state for the emission of fluorescence light with excitation light. The fraction of the molecules in the active state is kept so small that the fluorescence light from the sample may be assigned to individual molecules of the fluorescent dye. Thus, via the relative intensity distribution of the fluorescence light from one individual fluorescent molecule over several pixels of a two-dimensional detector array recording the fluorescence light, the position of the fluorescent molecule in the sample may be determined at a spatial resolution beyond the diffraction limit. Here, however, it is a precondition that a number of fluorescence light photons emitted by the individual fluorescent molecule which is sufficient from a statistical point of view reaches the detector. This does not only prolong the measurement time but also requires that the fluorescent dye molecules are at all suited for emitting a larger number of fluorescence light photons before they return in their non-active state or before they are transferred into a further state in which they are not excited for fluorescence by the excitation light. In this way, the number of practically available activatable fluorescent dyes which may in principle be the same switchable fluorescent dyes which are used in the RESOLFT technique is strongly limited.
A technique known as GSDIM is inverse as compared to PALM and may be applied with common fluorescent dyes. The inversion means that so many molecules of the fluorescent dye are temporarily deactivated into a dark state in GSDIM that the remaining fluorescent dye molecules are detectable individually. In this technique, however, the molecules of the fluorescent dye are all subjected to high light intensities and thus, prior to their actual measurement, to a correspondingly high number of changes of state to transfer them into their dark state and to keep their majority in this dark state.
DE 103 25 459 A1 discloses a method of forming a spatial structure at a spatial resolution beyond the diffraction limit. In this method, a substance is only left in a reactive state in a writing area which corresponds to an intensity minimum of an optical signal. Outside the writing area, the substance is transferred into a non-reactive state by means of the optical signal. When the substance so far as being in its reactive state is converted by means of a physical signal, this conversion is limited to the writing area. Also in this case, the substance is already subjected to very high light intensities and correspondingly many changes of state within short time when the writing area approaches a certain point of a substrate.
A further method of determining the distribution of a substance in a measurement region and a scanning light microscope are known from DE 10 1005 034 443 A1. Here, a luminescent sample is only subjected to excitation radiation of a single wavelength to avoid the effort associated with other methods for enhancing the spatial resolution. By means of the excitation radiation, the sample is transferred out of a first luminescence state in which the excitation for emission of luminescence radiation increases with increasing excitation radiation power up to a maximum value which corresponds to an excitation radiation power threshold value into a second luminescence state in which the sample displays a reduced excitability for emission of the luminescence radiation as compared to the first state. By irradiating the excitation radiation at a power above the threshold value the sample is transferred into the second state. In that the irradiation of excitation radiation takes place with an excitation radiation distribution which comprises a local power maximum above the threshold value and a local power minimum below the threshold value, the sample is transferred into the first state within partial areas and into the second state within adjacent partial areas. As a result, an image of the luminescent sample comprises sample areas in the first state and sample areas in the second state wherein predominantly sample areas in the first state contribute to the image of the luminescent sample and wherein the image has an increased spatial resolution as compared to the excitation radiation distribution. Particularly, the sample may be illuminated with the excitation radiation being focused to a line, wherein the power of the excitation radiation is, for example, sinusoidally modulated along the line so that the power is above the threshold value in some line sections and below the threshold value in some other line sections. Then, a scanning movement is made perpendicular to and along the line; and a detector for the luminescence radiation is a suitable high resolution line detector. The known method and the known microscope are depending on luminescent substances which comprise the described behavior with regard to an increasing excitation radiation power at a single wavelength. This is neither the case for all fluorescent dyes nor for all other luminescent substances nor for all so-called switchable fluorescence dyes, switchable fluorescent proteins and activatable luminescent particles.
There still is a need for a method of and a scanning light microscope for determining the distribution of a substance in a measurement region at a high spatial resolution in which fewer limitations with regard to the substances apply. Further, in a method of locally initiating a conversion of a substance in a conversion area, the substance, prior to initiating its conversion with a physical signal, should only be subjected to a low number of changes of state.